Novel algorithms for improved detection and analysis of fluorescent signal fluctuations

Stopper, Gebhard; Caudal, Laura C.; Rieder, Phillip; Gobbo, Davide; Stopper, Laura; Felix, Lisa; Everaerts, Katharina; Bai, Xianshu; Rose, Christine R.; Scheller, Anja; Kirchhoff, Frank

doi:10.1007/s00424-023-02855-3

Cited by 5 publications

(2 citation statements)

References 32 publications

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“…Cortical astrocytic Ca 2+ activity was recorded upon drug application. Analysis of Ca 2+ imaging data was performed using the custom-made detection and analysis software MATLAB (MSparkles v. 1.8.18)(Stopper et al, 2023) as previously described (Rieder et al, 2022). Shortly, fluorescence fluctuations at basal Ca 2+ concentrations (F 0 ) were computed along the temporal axes of each individual pixel using a polynomial fitting in a least-squares sense.…”

Section: Methodsmentioning

confidence: 99%

“…x 170 μm) from the cortical layers II-III of the SSp-ctx were chosen at a depth ranging from 30 to 100 μm from the slice surface and displaying a uniform astroglial distribution. Focal Analysis of Ca 2+ imaging data was performed using the custom-made detection and analysis software MATLAB (MSparkles v. 1.8.18) (Stopper et al, 2023) as previously described (Rieder et al, 2022). Shortly, fluorescence fluctuations at basal Ca 2+ concentrations (F 0 )…”

Section: Ca 2+ Imaging In Hippocampal Slicesmentioning

confidence: 99%

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Adenosine triggers early astrocyte reactivity that provokes microglial activation and drives the pathogenesis of sepsis-associated encephalopathy

Guo,

Gobbo,

Zhao

et al. 2023

Preprint

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SummaryMolecular pathways mediating systemic inflammation entering the brain parenchyma to induce sepsis-associated encephalopathy (SAE) remain elusive. Here, we report that in mice during the first 6 hours of peripheral lipopolysaccharide (LPS)-evoked systemic inflammation (6 hpi), the plasma level of adenosine quickly increased and enhanced the tone of central extracellular adenosine which then provoked neuroinflammation by triggering early astrocyte reactivity. Specific ablation of astrocytic A1 adenosine receptors (A1ARs) prevented this early reactivity and reduced the levels of inflammatory factors (e.g., CCL2, CCL5, and CXCL1) in astrocytes, thereby alleviating microglial activation, ameliorating blood-brain barrier disruption, neuronal dysfunction, and depression-like behaviour in the mice. Chemogenetic stimulation of Gi signaling in A1AR-deficent astrocytes at 2 and 4 hpi of LPS injection could restore neuroinflammation and depression-like behaviour, highlighting astrocytes rather than microglia as early drivers of neuroinflammation. Our results identify early astrocyte reactivity towards peripheral and central levels of adenosine as a novel pathway driving SAE.

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Section: Methodsmentioning

confidence: 99%

Section: Ca 2+ Imaging In Hippocampal Slicesmentioning

confidence: 99%

Adenosine triggers early astrocyte reactivity that provokes microglial activation and drives the pathogenesis of sepsis-associated encephalopathy

Guo,

Gobbo,

Zhao

et al. 2023

Preprint

Self Cite

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Adenosine triggers early astrocyte reactivity that provokes microglial responses and drives the pathogenesis of sepsis-associated encephalopathy in mice

Guo,

Gobbo,

Zhao

et al. 2024

Nat Commun

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Molecular pathways mediating systemic inflammation entering the brain parenchyma to induce sepsis-associated encephalopathy (SAE) remain elusive. Here, we report that in mice during the first 6 hours of peripheral lipopolysaccharide (LPS)-evoked systemic inflammation (6 hpi), the plasma level of adenosine quickly increased and enhanced the tone of central extracellular adenosine which then provoked neuroinflammation by triggering early astrocyte reactivity. Specific ablation of astrocytic Gi protein-coupled A1 adenosine receptors (A1ARs) prevented this early reactivity and reduced the levels of inflammatory factors (e.g., CCL2, CCL5, and CXCL1) in astrocytes, thereby alleviating microglial reaction, ameliorating blood-brain barrier disruption, peripheral immune cell infiltration, neuronal dysfunction, and depression-like behaviour in the mice. Chemogenetic stimulation of Gi signaling in A1AR-deficent astrocytes at 2 and 4 hpi of LPS injection could restore neuroinflammation and depression-like behaviour, highlighting astrocytes rather than microglia as early drivers of neuroinflammation. Our results identify early astrocyte reactivity towards peripheral and central levels of adenosine as an important pathway driving SAE and highlight the potential of targeting A1ARs for therapeutic intervention.

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A Deep Learning-Based Segmentation of Cells and Analysis (DL-SCAN)

Bhattarai,

Meyer,

Petersilie

et al. 2024

Preprint

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With the recent surge in the development of highly selective probes, fluorescence microscopy has become one of the most widely used approaches to study cellular properties and signaling in living cells and tissues. Traditionally, microscopy image analysis heavily relies on manufacturer-supplied software, which often demands extensive training and lacks automation capabilities for handling diverse datasets. A critical challenge arises, if fluorophores employed exhibit low brightness and low Signal-to-Noise ratio (SNR). As a consequence, manual intervention may become a necessity, introducing variability in the analysis outcomes even for identical samples when analyzed by different users. This leads to the incorporation of blinded analysis which ensures that the outcome is free from user bias to a certain extent but is extremely time-consuming. To overcome these issues, we have developed a tool called DL-SCAN that automatically segments and analyzes fluorophore-stained regions of interest such as cell bodies in fluorescence microscopy images using a Deep Learning algorithm called Stardist. We demonstrate its ability to automate cell identification and study cellular ion dynamics using synthetic image stacks with varying SNR. This is followed by its application to experimental Na+ and Ca2+ imaging data from neurons and astrocytes in mouse brain tissue slices exposed to transient chemical ischemia. The results from DL-SCAN are consistent, reproducible, and free from user bias, allowing efficient and rapid analysis of experimental data in an objective manner. The open-source nature of the tool also provides room for modification and extension to analyze other forms of microscopy images specific to the dynamics of different ions in other cell types.

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Novel algorithms for improved detection and analysis of fluorescent signal fluctuations

Cited by 5 publications

References 32 publications

Adenosine triggers early astrocyte reactivity that provokes microglial activation and drives the pathogenesis of sepsis-associated encephalopathy

Adenosine triggers early astrocyte reactivity that provokes microglial activation and drives the pathogenesis of sepsis-associated encephalopathy

Adenosine triggers early astrocyte reactivity that provokes microglial responses and drives the pathogenesis of sepsis-associated encephalopathy in mice

A Deep Learning-Based Segmentation of Cells and Analysis (DL-SCAN)

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